Microbial Protein Binding to gC1qR Drives PLA2G1B-Induced CD4 T-Cell Anergy.

The origin of the impaired CD4 T-cell response and immunodeficiency of HIV-infected patients is still only partially understood. We recently demonstrated that PLA2G1B phospholipase synergizes with the HIV gp41 envelope protein in HIV viremic plasma to induce large abnormal membrane microdomains (aMMDs) that trap and inactivate physiological receptors, such as those for IL-7. However, the mechanism of regulation of PLA2G1B activity by the cofactor gp41 is not known. Here, we developed an assay to directly follow PLA2G1B enzymatic activity on CD4 T-cell membranes. We demonstrated that gp41 directly binds to PLA2G1B and increases PLA2G1B enzymatic activity on CD4 membrane. Furthermore, we show that the conserved 3S sequence of gp41, known to bind to the innate sensor gC1qR, increases PLA2G1B activity in a gC1qR-dependent manner using gC1qR KO cells. The critical role of the 3S motif and gC1qR in the inhibition of CD4 T-cell function by the PLA2G1B/cofactor system in HIV-infected patients led us to screen additional microbial proteins for 3S-like motifs and to study other proteins known to bind to the gC1qR to further investigate the role of the PLA2G1B/cofactor system in other infectious diseases and carcinogenesis. We have thus extended the PLA2G1B/cofactor system to HCV and Staphylococcus aureus infections and additional pathologies where microbial proteins with 3S-like motifs also increase PLA2G1B enzymatic activity. Notably, the bacteria Porphyromonas gingivalis, which is associated with pancreatic ductal adenocarcinoma (PDAC), encodes such a cofactor protein and increased PLA2G1B activity in PDAC patient plasma inhibits the CD4 response to IL-7. Our findings identify PLA2G1B/cofactor system as a CD4 T-cell inhibitor. It involves the gC1qR and disease-specific cofactors which are gC1qR-binding proteins that can contain 3S-like motifs. This mechanism involved in HIV-1 immunodeficiency could play a role in pancreatic cancer and several other diseases. These observations suggest that the PLA2G1B/cofactor system is a general CD4 T-cell inhibitor and pave the way for further studies to better understand the role of CD4 T-cell anergy in infectious diseases and tumor escape.

PLA2G1B is involved in CD4 anergy and CD4 lymphopenia in HIV-infected patients.

The precise mechanism leading to profound immunodeficiency of HIV-infected patients is still only partially understood. Here, we show that more than 80% of CD4+ T cells from HIV-infected patients have morphological abnormalities. Their membranes exhibited numerous large abnormal membrane microdomains (aMMDs), which trap and inactivate physiological receptors, such as that for IL-7. In patient plasma, we identified phospholipase A2 group IB (PLA2G1B) as the key molecule responsible for the formation of aMMDs. At physiological concentrations, PLA2G1B synergized with the HIV gp41 envelope protein, which appears to be a driver that targets PLA2G1B to the CD4+ T cell surface. The PLA2G1B/gp41 pair induced CD4+ T cell unresponsiveness (anergy). At high concentrations in vitro, PLA2G1B acted alone, independently of gp41, and inhibited the IL-2, IL-4, and IL-7 responses, as well as TCR-mediated activation and proliferation, of CD4+ T cells. PLA2G1B also decreased CD4+ T cell survival in vitro, likely playing a role in CD4 lymphopenia in conjunction with its induced IL-7 receptor defects. The effects on CD4+ T cell anergy could be blocked by a PLA2G1B-specific neutralizing mAb in vitro and in vivo. The PLA2G1B/gp41 pair constitutes what we believe is a new mechanism of immune dysfunction and a compelling target for boosting immune responses in HIV-infected patients.

Maternal age, infant age, feeding options, single/multiple pregnancy, type of twin sets and mother-to-child transmission of HIV.

BACKGROUND: Some risk factors for mother-to-child transmission (MTCT) of HIV have been identified. To further reduce MTCT, other risk factors were evaluated. MATERIALS AND METHODS: A retrospective study on early infant diagnosis was conducted. Two-sided chi-square test was used to assess associations with infant HIV status. RESULTS: A total of 15 233 HIV-infected mothers and 15 404 infants were recruited. MTCT rate was 9.34%. Only 3.8% of infants born to mothers on antiretroviral treatment were infected. Under nevirapine, 4.1% of infants were infected. MTCT increased with infant' age at testing. Younger mothers tend to transmit more HIV (P = 0.003). More children were infected in single pregnancies compared with multiple pregnancies, P < 0.001. There were more infections in male-female twins' sets (P = 0.037). CONCLUSIONS: Maternal age, type of pregnancy and twins' sets are new MTCT risk factors. Strategies to further decrease transmission through family planning, pre/post natal consultations and clinical practices are needed.

Altered responses to homeostatic cytokines in patients with idiopathic CD4 lymphocytopenia.

Idiopathic CD4 lymphocytopenia (ICL) is a rare immune deficiency characterized by a protracted CD4(+) T cell loss of unknown etiology and by the occurrence of opportunistic infections similar to those seen in AIDS. We investigated whether a defect in responses to cytokines that control CD4(+) T cell homeostasis could play a role in ICL. Immunophenotype and signaling responses to interleukin-7 (IL-7), IL-2, and thymic stromal lymphopoietin (TSLP) were analyzed by flow cytometry in CD4(+) T cells from 15 ICL patients and 15 healthy blood donors. The induction of phospho-STAT5 after IL-7 stimulation was decreased in memory CD4(+) T cells of some ICL patients, which correlated with a decreased expression of the IL-7Rα receptor chain (R = 0.74, p<0.005) and with lower CD4(+) T cell counts (R = 0.69, p<0.005). IL-2 responses were also impaired, both in the Treg and conventional memory subsets. Decreased IL-2 responses correlated with decreased IL-7 responses (R = 0.75, p<0.005), pointing to combined defects that may significantly perturb CD4(+) T cell homeostasis in a subset of ICL patients. Unexpectedly, responses to the IL-7-related cytokine TSLP were increased in ICL patients, while they remained barely detectable in healthy controls. TSLP responses correlated inversely with IL-7 responses (R = -0.41; p<0.05), suggesting a cross-regulation between the two cytokine systems. In conclusion, IL-7 and IL-2 signaling are impaired in ICL, which may account for the loss of CD4(+) T cell homeostasis. Increased TSLP responses point to a compensatory homeostatic mechanism that may mitigate defects in γc cytokine responses.

Short communication: nuclear JAK3 and its involvement in CD4 activation in HIV-infected patients.

The subcellular localization of JAK3 was examined by quantitative image analysis. For the first time, JAK3 was found to be located in the nuclei of primary CD4 lymphocytes. A comparable quantity of JAK3 was recovered in CD4 lymphocytes from healthy donors and HIV-infected patients. By contrast, far more phosphorylated JAK3 (pJAK3) was found in the nuclei of CD4 lymphocytes from HIV-infected patients than from healthy donors. The correlation detected between the quantity of pJAK3 in the nuclei of CD4 lymphocytes and the increase in HLA-DR at their surface suggests that pJAK3 may play a role in the deleterious immune activation characterizing HIV-infected patients.

Membrane microdomains and cytoskeleton organization shape and regulate the IL-7 receptor signalosome in human CD4 T-cells.

Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.

Characteristics of plasmacytoid dendritic cell and CD4+ T cell in HIV elite controllers.

Despite variability, the majority of HIV-1-infected individuals progress to AIDS characterized by high viral load and massive CD4+ T-cell depletion. However, there is a subset of HIV-1-positive individuals that does not progress and spontaneously maintains an undetectable viral load. This infrequent patient population is defined as HIV-1 controllers (HIV controllers), and represents less than 1% of HIV-1-infected patients. HIV-1-specific CD4+ T cells and the pool of central memory CD4+ T cells are also preserved despite immune activation due to HIV-1 infection. The majority of HIV controllers are also defined by the absence of massive CD4+ T-cell depletion, even after 10 years of infection. However, the mechanisms involved in protection against HIV-1 disease progression have not been elucidated yet. Controllers represent a heterogeneous population; we describe in this paper some common characteristics concerning innate immune response and CD4+ T cells of HIV controllers.

HIV controllers maintain a population of highly efficient Th1 effector cells in contrast to patients treated in the long term.

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.

Interleukin (IL)-2 and IL-15 have different effects on human natural killer lymphocytes.

Although interleukin (IL)-2 and IL-15 share the common signal transducing receptor chains IL-2Rß and γ(c) and give rise to the same signaling patterns in human natural killer (NK) cells in vitro, they differ in their effects on the development, activation, and proliferation of these cells in vivo. We have previously demonstrated that the activation of NK cells induces a cellular program characterized by the sequential transcription-regulated expression of IL-15 and IL-2 high-affinity receptors. We demonstrate here that these receptors induce different responses. IL-15 sustains the expression of its high-affinity receptor, leading to long-lasting STAT5 phosphorylation and BCL2 expression. By contrast, IL-2 induces the rapid disappearance of IL-2Rα and γ(c) chains when the gene transcription is downregulated, shutting down IL-2-responses as demonstrated by the absence of STAT5 phosphorylation and BCL2 expression.

HIV controllers: a multifactorial phenotype of spontaneous viral suppression.

A small minority of HIV-infected individuals, known as HIV controllers, is able to exert long-term control over HIV replication in the absence of treatment. Increasing evidence suggests that the adaptive immune system plays a critical role in this control but also that a combination of several host and/or viral factors, rather than a single cause, leads to this rare phenotype. Here, we review recent advances in the study of these remarkable individuals. We summarize the epidemiology and clinical characteristics of HIV controllers, and subsequently describe contributing roles of host genetic factors, innate and adaptive immune responses, and viral factors to this phenotype. We emphasize distinctive characteristics of HIV-specific CD4 T cell responses and of CD4 T cell subpopulations that are frequently found in HIV controllers. We discuss major controversies in the field and the relevance of the study of HIV controllers for the development of novel therapeutic strategies and vaccines.

HIV infection perturbs interleukin-7 signaling at the step of STAT5 nuclear relocalization.

OBJECTIVE: Interleukin-7 (IL-7) responses are impaired in CD4(+) T cells from HIV-infected patients, which may play a significant role in the loss of CD4(+) T-cell homeostasis. We set to investigate the nature of IL-7-dependent signaling defects in patients with progressive HIV-1 infection. DESIGN AND METHODS: IL-7 signaling was compared in CD4(+) T cells from viremic patients with a viral load more than 10,000 copies of HIV RNA/ml (n = 23) and from healthy blood donors (n = 23). Phosphorylation of the transcription factor STAT5 on the regulatory serine S726 and the key tyrosine Y694 was monitored by intracellular flow cytometry. Phospho-STAT5 relocalization to the nucleus was analyzed by quantitative immunofluorescence imaging. RESULTS: In control CD4(+) T cells, S726 phosphorylation was mostly constitutive and inducible by IL-7 to a limited extent (1.3x, P < 0.05). In contrast, phosphorylation at Y694 was highly inducible by IL-7 (12.6x, P < 0.0001). Progressive HIV infection led to hyperphosphorylation of both S726 and Y694 in naive CD4(+) T cells, with these changes correlating together (R = 0.66, P = 0.01). Quantitative image analysis revealed an impairment in the nuclear relocalization of both forms of phospho-STAT5 in patient cells (P < 0.005 for S726; P < 0.05 for Y694). The nuclear relocalization defect correlated with increased HLA-DR expression (R = 0.75, P < 0.01), suggesting a role for chronic immune activation in perturbed IL-7 signal transduction. CONCLUSION: HIV infection perturbs IL-7 signaling by impairing the access of STAT5 to the nuclear compartment. This defect may contribute to the loss of CD4(+) T-cell populations in patients with chronically high immune activation.

Efficacy of rifampin-moxifloxacin-metronidazole combination therapy in hidradenitis suppurativa.

BACKGROUND: Antibiotics have been shown to improve hidradenitis suppurativa (HS) patients but complete remission is rare using these treatments. OBJECTIVE: To assess the efficacy and safety of a combination of oral rifampin, moxifloxacin and metronidazole in long-lasting refractory HS. METHODS: We retrospectively studied 28 consecutive HS patients including 6, 10 and 12 Hurley stage 1, 2 and 3 patients, respectively. Complete remission, defined as a clearance of all inflammatory lesions including hypertrophic scars, was the main outcome criterion of the study. RESULTS: Complete remission was obtained in 16 patients, including 6/6, 8/10 and 2/12 patients with Hurley stage 1, 2 and 3, respectively (p=0.0004). The median duration of treatment to obtain complete remission was 2.4 (range 0.9-6.5) and 3.8 months (range 1.6-7.4) in stage 1 and 2 patients, respectively, and 6.2 and 12 months in the 2 stage 3 patients. Main adverse events of the treatments were gastrointestinal disorders (64% of patients) and vaginal candidiasis (35% of females). Reversible tendinopathy and hepatitis occurred in 4 and 1 patient, respectively. CONCLUSIONS: Complete remission of refractory HS can be obtained using broad-spectrum antibiotics and Hurley staging is a prognostic factor of response to the treatment.

[Role of CD4 lymphocytes in the efficient immune response of HIV controllers]. / Les patients" VIH contrôleurs ": rôle des lymphocytes T CD4 dans la stimulation d'une réponse immunitaire efficace.

More than 30 million people are infected by HIV worldwide. The rate of progression to AIDS is variable. A newly identified category of patients, so-called HIV controllers, spontaneously control HIV replication for long periods. HIV controllers are defined as infected patients whose viral load remains below 400 RNA copies/ml of blood, without treatment, for at least five years. We have studied the immune system of these patients, and particularly their CD4 lymphocytes, that participate in the induction, intensity, quality and duration of immune responses. CD4 lymphocytes in patients who progress to AIDS are directly targeted by the virus and participate in the activation that leads to immune deficiency. Central memory CD4 cells exhibit unusual properties in HIV controllers. Apart from the fact that they persist in large numbers and secrete large amounts of interleukin-2, which serves as an autocrine factor for their own growth, central memory CD4 cells overexpress the CCR7 homing molecule that allows them to leave capillaries and rapidly enter lymph nodes, the site of effective immune responses. These CD4 lymphocytes also men multiply rapidly, and some of them express receptors with very high antigen avidity. The presence of these central memory CD4 cells and their characteristic properties may explain the remarkable stability observed in HIV controllers. We suspect that these lymphocytes react very rapidly and efficiently to any breakthrough in viral replication. The putative relationship between HIV controllers and patients who remain asymptomatic and uninfected despite repeated exposure to the virus is discussed. Our new findings should help to guide antiretroviral therapy and vaccination strategies.

IL-2 induces conformational changes in its preassembled receptor core, which then migrates in lipid raft and binds to the cytoskeleton meshwork.

While interleukin (IL)-2 clearly initiates the sequential assembly of its soluble receptor fragments (sIL-2R) in vitro (with sIL-2Rα first, sIL-2Rß second, and sγc last), the assembly mechanism of full-length subunits (IL-2R) at the surface of living lymphocytes remains to be elucidated. Here we demonstrate by fluorescence cross-correlated spectroscopy that native IL-2Rß and γc assemble spontaneously at the surface of living human leukemia T cells (Kit-225 cell line) in the absence of IL-2 and with 1:1 stoichiometry. The dissociation constant of the membrane-embedded IL-2Rß/γc complex is measured in situ. Förster fluorescence resonance energy transfer analyzed by confocal microscopy of transfected COS-7 cells between combination pairs of various-length receptor chain constructions, using green fluorescent protein derivatives as cytoplasmic carboxy-terminal extensions, showed that IL-2Rß:ECFP and γc:EYFP bind each other through their extracellular domains, and that IL-2 binding brings their transmembrane domains 30 Å closer together. These observations demonstrate that IL-2Rß/γc heterodimers are preformed and that their cytoplasmic domains, carrying Janus kinase (Jak) 1 and Jak3, are pulled and tethered together on cytokine binding, triggering signaling transduction. IL-2 binding stabilizes IL-2/IL-2R complexes in membrane nanodomains that promote Jak1/Jak3 phosphorylation. The complexes then interact with the cytoskeleton, which slows receptor diffusion (as measured by fluorescence cross-correlated spectroscopy) and promotes STAT (signal transducer and activator of transcription) 5 phosphorylation. Separation of IL-2-activated receptors from Triton-lysed cells in detergent-resistant membrane nanodomains by ultracentrifugation on a sucrose gradient confirmed their presence in lipid rafts. The release of the IL-2-activated receptor from cytochalasin-treated cells and the IL-2-induced recruitment of actin and tubulin, analyzed by immunoprecipitation, confirmed that the activated receptor interacts with the cytoskeleton. Although IL-2Rα (the third chain that gives the IL-2Rß/γc receptor core its high affinity for IL-2) is highly expressed at the cell surface and mainly clustered in membrane microdomains at the surface of Kit-225 cells, the few free IL-2Rα present bind last to the IL-2/IL-2Rß/γc complex and lock IL-2 to its binding site for prolonged action, promoting signal amplification.

HIV controller CD4+ T cells respond to minimal amounts of Gag antigen due to high TCR avidity.

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vbeta repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-gamma responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vbeta repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia.

Interleukin-7 compartmentalizes its receptor signaling complex to initiate CD4 T lymphocyte response.

Interleukin (IL)-7 is a central cytokine that controls homeostasis of the CD4 T lymphocyte pool. Here we show on human primary cells that IL-7 binds to preassembled receptors made up of proprietary chain IL-7Ralpha and the common chain gammac shared with IL-2, -4, -9, -15, and -21 receptors. Upon IL-7 binding, both chains are driven in cholesterol- and sphingomyelin-rich rafts where associated signaling proteins Jak1, Jak3, STAT1, -3, and -5 are found to be phosphorylated. Meanwhile the IL-7.IL-7R complex interacts with the cytoskeleton that halts its diffusion as measured by single molecule fluorescence autocorrelated spectroscopy monitored by microimaging. Comparative immunoprecipitations of IL-7Ralpha signaling complex from non-stimulated and IL-7-stimulated cells confirmed recruitment of proteins such as STATs, but many others were also identified by mass spectrometry from two-dimensional gels. Among recruited proteins, two-thirds are involved in cytoskeleton and raft formation. Thus, early events leading to IL-7 signal transduction involve its receptor compartmentalization into membrane nanodomains and cytoskeleton recruitment.

Dual mechanism of impairment of interleukin-7 (IL-7) responses in human immunodeficiency virus infection: decreased IL-7 binding and abnormal activation of the JAK/STAT5 pathway.

Interleukin-7 (IL-7) plays a central role in controlling the homeostasis of both naive and long-term-memory CD4(+) T cells. To better understand how human immunodeficiency virus (HIV) perturbs CD4(+) T-cell homeostasis, we performed a detailed analysis of IL-7R expression, IL-7 binding, and IL-7-dependent early and late signaling events in CD4(+) T-cell subsets from viremic and efficiently treated patients. HIV infection differentially affected the expression of IL-7 receptor (IL-7R) chains, with decreases in IL-7Ralpha/CD127 expression in the memory subset and increases in gammac/CD132 expression in all CD4(+) T cells. This resulted in preserved IL-7 binding in the naive compartment and decreased IL-7 binding in the memory compartment of viremic patients. Accordingly, the percentages of cells signaling in response to IL-7, as measured by pSTAT5 induction, were decreased in memory subsets, including conventional CD4(+) T cells and regulatory T cells. However, the levels of pSTAT5 induction per responding cell, as measured by pSTAT5 fluorescence intensity, were increased within all naive and memory CD4(+) T-cell subsets of viremic patients. The basal level of pSTAT5 was also increased, indicating a constitutive activation of the JAK/STAT5 pathway. IL-7 functional responses, as measured by Bcl-2, CD25, and Foxp3 induction, were impaired in viremic patient CD4(+) T cells, suggesting that chronic activation led to downstream defects in the STAT5 signaling pathway. Thus, HIV infection perturbs IL-7 responses at both receptor binding and signaling steps, which likely compromises the regenerative capacity of the CD4(+) T-cell pool and may contribute to CD4(+) T-cell depletion.